Deciphering cell states and genealogies of human haematopoiesis

The human blood system is maintained through the differentiation and massive amplification of a limited number of long-lived haematopoietic stem cells (HSCs)1. Perturbations to this process underlie diverse diseases, but the clonal contributions to human haematopoiesis and how this changes with age remain incompletely understood. Although recent insights have emerged from barcoding studies in model systems2–5, simultaneous detection of cell states and phylogenies from natural barcodes in humans remains challenging. Here we introduce an improved, single-cell lineage-tracing system based on deep detection of naturally occurring mitochondrial DNA mutations with simultaneous readout of transcriptional states and chromatin accessibility. We use this system to define the clonal architecture of HSCs and map the physiological state and output of clones. We uncover functional heterogeneity in HSC clones, which is stable over months and manifests as both differences in total HSC output and biases towards the production of different mature cell types. We also find that the diversity of HSC clones decreases markedly with age, leading to an oligoclonal structure with multiple distinct clonal expansions. Our study thus provides a clonally resolved and cell-state-aware atlas of human haematopoiesis at single-cell resolution, showing an unappreciated functional diversity of human HSC clones and, more broadly, paving the way for refined studies of clonal dynamics across a range of tissues in human health and disease.


Introduction
Protocol for single-cell Regulatory multi-omics with Deep Mitochondrial mutation profiling.Following this protocol, three separate libraries plus one optional library (mtDNA lib; RNA lib, ATAC lib, and an optional Hashing library) will be generated in parallel for joint single-cell profiling.This protocol has been tested for hematopoietic stem/progenitor and differentiated cells (Including T cells, B cells, NK cells, monocyte, dendritic cells, plasma, GMP, MDP, CLP, MPP, HSC, etc).For other tissues or cell types, some conditions may need to be further optimized.
. Add 5 μl Human TruStain FcX™ in each sample, mix and incubate at 4°C for 10 minutes to for blocking.
. During the incubation, for each sample, dilute 1 μl TotalSeqTM antibody (unique for each sample) in 50 μl FACS buffer in a corresponding fresh, 1.5 ml, low-binding tube .Transfer the corresponding 50 ul antibody dilution from step 4 into each of the blocked-cell suspensions, and incubate at 4°C for 30 minutes.
. Add 3 ml FACS buffer in each sample and centrifuge 500g at 4°C for 5 minutes for washing.
. Remove supernatant and repeat the wash 1 more time with 3ml FACS buffer for each sample and centrifuge 500g 4°C for 5 min .Pool Hashed samples together (usually equal cell number) as one sample to proceed forward.

Fixation and permeabilization
We found that decreasing BSA concentration in most of the buffers can improve the mtDNA recovery rate.The in-house buffers provided in Material section are optimized for hematopoietic cells.The recipes can be further optimized for different tissues and cell types.
. For each sample, resuspend cells in 1ml Fixation buffer, and incubate at room temperature for 10 minutes.
. Quench the fixation reaction with 50 ul glycine (2.5M) at final concentration of 0.125M, and incubate at room temperature for 3 minutes.
. Remove supernatant, add 1ml Perm buffer, and incubate for 5 minutes on ice.Then immediately add Perm-Wash buffer 3ml, and centrifuge 500g at 4°C for 5 minutes.
. Remove the top of supernatant without disturbing cell pellet, leaving 20~100 ul Resuspension buffer, and resuspend and count the cells.Ideally, make the cell concentration equal to or more than 2800 cell/ul

Tagmentation with GDN
We found that adding GDN during tagmentation can increase the mtDNA recovery rate without significantly affecting RNA and ATAC quality.But standard tagmentation reaction without GDN also works to proceed forward.
. Generate the following mix per sample for tagmentation:
ATAC  .Purify the PCR product.
. Run Bioanalyzer for the qPCR product using Agilent High Sensitivity DNA Kit.
. The product from step 25 is also ready for mitochondrial DNA capture.Typically, 2000 ng is needed.
Mitochondrial DNA (mtDNA) library prep .For each sample, prepare 500ng ATAC library X4 (2000ng in total) for four hybridization reactions using staggered probes (mitoV1, mitoV2, mitoV3, mitoV4) . The hybridization is performed by following IDT xGen™ hybridization capture guide.The updating protocol can be found on IDT website.Briefly, the capture is a two-day protocol as shown below. .Combine every four wells for each sample (corresponding to 4 hybridizations using probes mitoV1, mitoV2, mitoV3, mitoV4) in total 200ul and purify by 1.6X SPRI beads (320 ul SPRI each), finally elute in 25 ul ddH2O.
. Run Bioanalyzer for the purified PCR product using Agilent High Sensitivity DNA Kit.The expected mtDNA fragment trace look like below:
(Optional) Hashing library .If cell hashing was performed, the hashing library is prepared using PCR as below, which is also monitored by
Go to step 2 for 35 more cycles.Stop the qPCR manually based on the amplification curve.Usually around 10 or below 10 cycles when the curve is reaching plateau. .Run Bioanalyzer for the qPCR product using Agilent High Sensitivity DNA Kit.The Expected ATAC traces are as below.Different cell types may show slightly different trace.ATAC library prep (Scale-up).Scale up the PCR above to 8 wells per sample, (50 μL per well, for a total 400 μL PCR mix), monitored using SYBR Green.Each well should contain:the same program in small-scale test: 1. 98 °C 45s; 2. 98 °C 10s; 3. 63 °C 30s; 4. 72 °C 60s; 5.Read Plate; 6. Go to step 2 for 35 more cycles.Stop the qPCR manually based on the amplification curve.Limit the number of cycles to 10 or lower.

. D1 :
Setting up hybridization reaction following APPENDIX A in xGen™ hybridization capture guide.xGen Hyb Panel is replaced by one of the probes for each reaction: mitoV1/mitoV2/mitoV3/mitoV4.The hybridization reaction is as below: is performed 95 °C 30s, followed by 65 °C 16h .D2: Capture and washes are performed following Tube protocol xGen™ hybridization capture guide (from Page 23).The updating protocol can be found on IDT website .The post-capture PCR is monitored by SYBR: 1 well of PCR (as below) is performed for each the following program: 1. 98 °C 45s; 2. 98 °C 10s; 3. 63 °C 30s; 4. 72 °C 20s; 5. Read Plate; 6. Go to step 2 for 35 more cycles.Stop the qPCR manually based on the amplification curve.Limit the number of cycles to 3-5 cycles.